Inhibition of PKB improves engraftment levels in β2-microglobulin^−/− NOD/SCID mice. (A) CD34⁺ cells, cultured in the presence of SCF and FLT-3L, were transduced with empty vector alone or myrPKB. One day after transduction, cells were injected into β2-microglobulin^−/− NOD/SCID mice. Six weeks after injection, mice were killed, and the percentage of eGFP⁺ cells in the human CD45 population in the bone marrow was determined. Data were depicted as the ratio between the percentage of eGFP⁺ cells in the human population before and after migration. A indicates after; and B, before. (B) CD34⁺ cells were cultured in the absence or presence of the PKB inhibitor VIII for 48 hours, on which cells were injected into β2-microglobulin^−/− NOD/SCID mice from 3 different cages (1, 2, and 3). For 3-week experiments, 348 000 cells were injected, whereas for the 6-week experiments 36 166 cells were injected. Six weeks after injection, mice were killed, and the percentage of human CD45⁺ cells in the bone marrow was determined by flow cytometric analysis. (C) Data were depicted as the fold induction of the percentage of human CD45⁺ cells in the bone marrow of mice that received a with cells pretreated with the PKB inhibitor compared with controls as a combination of results from mice killed 3 and 6 weeks after transplantation (n = 4). (D) Data were depicted as the percentage of human CD45⁺ cells in the bone marrow. (E) Flow count beads were used to calculate the absolute numbers of human cells in the bone marrow. (F-J) Magnetic separation was performed to separate the human CD45⁺ population from the mouse bone marrow. (F-H) Flow cytometric analysis was subsequently performed to determine the percentage of (F) CD34⁺, (G) CD19⁺, and (H) CD235a⁺ cells in the human population. (I-J) In addition, cytospins were made to determine the percentage of eosinophils and neutrophils in the human population. 7-AAD indicates 7-amino-actinomycin D. Error bars represent SEM. Samples significantly different (P < .05) are indicated with horizontal lines and asterisks.